Fischell Department of Bioengineering Theses and Dissertations

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3D ENGINEERING OF VIRUS-BASED PROTEIN NANOTUBES AND RODS: A TOOLKIT FOR GENERATING NOVEL NANOSTRUCTURED MATERIALS
(2018) Brown, Adam Degen; Culver, James N; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Technological innovation at the nanometer scale has the potential to improve a wide range of applications, including energy storage, sensing of environmental and medical signals, and targeted drug delivery. A key challenge in this area is the ability to create complex structures at the nanometer scale. Difficulties in meeting this challenge using traditional fabrication methods have prompted interest in biological processes, which provide inspiration for complex structural organization at nanometer to micrometer length scales from self-assembling components produced inexpensively from common materials. From that perspective, a system of targeted modifications to the primary amino acid structure of Tobacco mosaic virus (TMV) capsid protein (CP) has been developed that induces new self-assembling behaviors to produce nanometer-scale particles with novel architectures. TMV CPs contain several negatively charged carboxylate residues which interact repulsively with those of adjacent CP subunits to destabilize the assembled TMV particle. Here, the replacement of these negatively charged carboxylate residues with neutrally charged or positively charged residues results in the spontaneous assembly of bacterially expressed CP into TMV virus-like particles (VLPs) with a range of environmental stabilities and morphologies and which can be engineered to attach perpendicularly to surfaces and to display functional molecular patterns such as target-binding peptide chains or chemical groups for attachment of functional targets. In addition, the distinct electrostatic surface charges of these CP variants enable the higher-level coassembly of TMV and VLP into continuous rod-shaped nanoparticles with longitudinally segregated distribution of functionalities and surface properties. Furthermore, the unique, novel, environmentally responsive assembly and disassembly behaviors of the modified CPs are shown to act as simple mechanisms to control the fabrication of these hierarchically structured functional nanoparticles.
3D PRINTED MULTILAYERED / INTERFACIAL SCAFFOLDS FOR OSTEOCHONDRAL REGENERATION
(2021) choe, Robert; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Osteoarthritis is a highly prevalent rheumatic musculoskeletal disorder that primarily affects the knee joint. This disease is characterized by the progressive breakdown of the articular cartilage and remodeling of the subchondral bone in the synovial joint. Repetitive overloading perpetuates the damage to the affected cartilage and undermines the structural integrity of the osteochondral unit. Despite much research in osteochondral tissue engineering, no particular strategy has stood out as a potential alternative to conventional treatment options. One major issue that arises during osteochondral regeneration is that the defect site is exposed to a significant physiological load. To overcome these challenges, various tissue engineering strategies have been employed to design multiphasic osteochondral scaffolds that recapitulate layer-specific biomechanical properties. However, multilayered scaffolds have failed to fully satisfy the mechanical requirements to persists within the osteochondral defect. Through the use of extrusion-based bioprinting, we attempt to fabricate a biphasic osteochondral scaffold with improved load-bearing properties and a mechanically strong interface.
Acquired Platelet And Neutrophil Dysfunction Due To High Mechanical Shear Stress
(2022) Arias, Katherin; Wu, Zhongjun; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Heart failure (HF) is a public health burden. In the next ten years, 8 million Americans are expected to have HF. A subset of these patients will develop advanced HF. They are refractory to medical therapies and have limited treatment options, including heart transplantation or a left ventricular assist device (LVAD). Heart transplants for all advanced HF patients are impractical due to the scarcity of donors. LVAD therapy is the sole viable option for advanced HF patients as a bridge to transplant, a temporary treatment while the heart recovers, and a long-term destination therapy. Over the last two decades, significant progress in LVADs have been made through various iterations. Advances in LVADs have been due to redesign focused on lowering adverse events. However, bleeding and infections are still the most prevalent adverse complications. LVADs and other mechanical circulatory support devices induce damage to blood cells and plasma components due to the high mechanical shear stress (HMSS) generated. Therefore, there must be a link between LVAD-induced cellular damage and the adverse events experienced in LVAD patients. This dissertation aimed to investigate the relationship between cellular blood damage and LVAD-associated complications and qualify the extent of cellular damage/defects and functional alterations.The overall objective of this dissertation was to investigate the acquired cellular defects of platelets and neutrophils in blood after shear stress exposure. This objective was accomplished through in-vivo, in-vitro, and in-silico studies. The in-vivo studies examined the shear stress-induced injury of platelets in LVAD recipients and linked the adverse bleeding events (Chapter 3). The in-vitro studies explored the shear stress-induced injury of leukocytes (Chapter 4). The extent of the structural damage and functional alterations related to shear stress level and the exposure time was quantified (Chapter 5 and Chapter 6). Finally, the in-silico studies developed a simulation of leukocyte function with experimental data that was used to predict the extent of the shear stress-induced leukocyte function change (Chapter 6). The damaging effects of the high shear stress produced by mechanical circulatory support devices such as LVADs were conveyed through an integrated biological and engineering approach.
ACTUATION OF MULTIFUNCTIONAL HARD NANOPARTICLES FOR ACTIVELY CONTROLLED DRUG RELEASE
(2019) Sangtani, Ajmeeta; Delehanty, James B; Stroka, Kimberly M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Systemic drug delivery relies on repeated dosing of large concentrations of poorly targeted drug leading to off-target toxicity. Recently, nanoparticle (NP)-mediated drug delivery (NMDD) has been developed as an approach to overcome the limitations of traditional drug delivery. The unique size-dependent properties of NPs and their ability to augment the activity of attached/loaded cargos makes them attractive drug delivery vectors. NPs are classified into two categories (soft or hard depending on their material composition) and our understanding of how to load and control soft NP materials currently surpasses that of hard NPs. In this dissertation we seek to further our fundamental knowledge of hard NP-based drug delivery systems. In Aim 1 we utilize a quantum dot (QD)-cell uptake peptide complex as a central scaffold to append various responsive peptide-drug constructs in order to modulate the toxicity of one of the most widely used chemotherapeutics, doxorubicin. By doing a comparative study of four chemical linkages, we determine the role played by attachment chemistry in controlling drug release. In Aim 2, we utilize the knowledge gained from Aim 1 to develop a system capable of overcoming multidrug resistance in cancer cells, which is known to severely limit the efficacy of chemotherapeutics. Our hard NP conjugate system is unique as it is one of the few systems reported in the literature to bypass multidrug resistance pumps without the need for exogenous drugs. Finally, in Aim 3 we append a peptide for membrane targeting and a photosensitizing drug capable of generating reactive oxygen species to the QD. This multifunctional system displays augmented therapeutic efficacy of the appended photosensitizer by delivering it to the membrane of cells and controlling its actuation using energy transfer. The work described here details basic concepts for the design of “smart” hard NP materials for internally and externally-triggered, active release of surface-appended drug cargos. Additionally, we hope to elucidate the important design considerations that must be taken into account when designing hard NP systems for controlled drug delivery.
Additive Manufacturing for Recapitulating Biology in vitro and Establishing Cellular & Molecular Communication
(2023) Chen, Chen-Yu; Bentley, William E.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Recapitulating biological systems within laboratory devices, particularly those with analytical instrumentation, has enhanced our ability to understand biology. Especially useful are systems that provide data at the length and time scales characteristic of the assembled biological systems. In this dissertation, we have employed two advanced technologies — additive manufacturing and electrobiofabrication to create systems that both recapitulate biology and provide ready access to molecular data. First, we utilized two-photon direct laser writing (DLW) and digital light processing (DLP) 3D printing to reconstruct morphologies of human gut villi. Our constructs enable small molecule diffusion through pores and enable epithelial cell growth and differentiation, as in the gastrointestinal (GI) tract. We also developed a cell/particle alignment methodology that applies a vacuum on the underside of a device to rapidly facilitate attachment to 3D printed scaffolds. These simple demonstrations of additive manufacturing show how one can better tailor geometric features of organ-on-a-chip and other in vitro models. We then added electrobiofabrication as a means create functionalized surfaces that rapidly assemble biological components, noted for their labile nature, onto devices with just an applied voltage. In one example, we show how a thiolated polyethylene glycol (PEG) can be electroassembled as a sensor interface that includes antibody binding proteins for both titer and glycan analysis. Rapid assessment of titer and glycan structure is important for biopharmaceuticals development and manufacture. While the interface and sensing methodology was performed using standard laboratory instrumentation, we show that the methodology can be streamlined and operated in parallel by incorporating into a microfluidic sensor platform. Additionally, we show how the combination of optical and electrochemical (redox) based measurements can be combined in a simplified insert that “fits” nearly any microplate reader or other fairly standardized laboratory spectrophotometric unit. We believe that by adapting transformative electrochemical analytical methods so they can augment more traditional optical techniques, we might ultimately generate devices that provide a far more comprehensive picture of the target, promoting better investigation. Specifically, we show how three important biological and chemical systems can be interrogated using both optical measurements and electrochemistry: the oxidation state of proteins including monoclonal antibodies, redox status of hydrogel materials, and electrobiofabrication and electrogenetic induction. Lastly, we demonstrate how electrobiofabrication can be used to create designer communities of bacteria — artificial biofilms — the study of which is important for understanding phenomena from infectious disease to food contamination. That is, we discovered that by varying the applied voltage, surface area, and composition of the to-be-assembled hydrogel solution, we can precisely control the intercellular environment among bacterial populations. In sum, this dissertation integrates advances in assembly, through additive manufacturing, electrobiofabrication, with advances in electrochemical analysis to bring to the fore an electronic understanding of complex biological phenomena. We believe that the capability of translating biological information into a processible digital language opens tremendous opportunities for advancing our understanding of nature’s amazing systems, potentially enabling electronic means to control her subsystems.
ADVANCED VISION INTELLIGENT METHODS FOR FOOD, AGRICULTURAL, AND HEALTHCARE APPLICATIONS
(2021) Wang, Dongyi; Tao, Yang; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
With fast software and hardware developments, vision intelligence models have attracted great attention and showed unprecedented performance on large-scale datasets. In practice, studies are still needed to design innovative intelligence models for niche applications with limited data accessibilities in uncertain real-world scenarios. This research casts light on cutting edge vision intelligent applications that enhance the essential areas of people’s livelihood, including food, agriculture, and healthcare. First, a 2D/3D imaging system was developed to facilitate the autonomous processing of Chesapeake Bay blue crabs, as an efficient solution to current hand-picking protocols. The system integrates a semantic segmentation model to understand crab 2D morphology. It can detect crab back-fin knuckles with R2 larger than 0.995, which guides movements of a two degree-of-freedom gantry station in removing crab legs and extracting crab body cores with 2mm accuracy. The customized active laser line scanning 3D range imaging system shows high imaging accuracy (0.15mm) and is able to assist a linear actuator in removing crab chamber cartilages. Second, computer aided vision intelligent methods were applied to an emerging ophthalmologic imaging modality known as, erythrocyte mediated angiography. A novel regression-based segmentation model and a Monte Carlo based tracking method were proposed to monitor the erythrocytes in stasis and in movements. Both models displayed comparable performance to human experts. Preliminary clinical results also manifest the potential relationships between paused erythrocyte densities and primary open-angle glaucoma. To better understand retinal vessel and erythrocyte distributions, a novel network architecture, the Hard Attention Net was proposed. This network has achieved state-of-art retinal vessel segmentation performance across different ophthalmologic imaging modalities. Finally, deep learning based qualitative and quantitative analyses were applied to spectral signals for monitoring high-level status and low-level chemical properties of agricultural bioproducts. Experiments include early-stage tomato spotted wilt virus detection as well as nutrition content estimation of plant and corn kernels. By using adversarial training and feature weighting ideas, the two proposed networks were effectively trained with a limited dataset. The results of these studies show great potential for vision intelligence models for promoting applications of advanced imaging modalities and vision-guided automations in food, agricultural, and healthcare fields.
ARRHYTHMOGENESIS AND CONDUCTION PROPERTIES OF CARDIOMYOCYTES IN RESPONSE TO DYSSYNCHRONOUS MECHANICAL AND ELECTRICAL STIMULATION
(2010) Chan, Dulciana; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Many cardiac therapeutic modalities, including pacemakers, implantable cardioverter defibrillators, and cardiac resynchronization therapy devices, are used to treat abnormalities in cardiac function and conduction. Both electrical and mechanical dyssynchrony can have deleterious effects including reduced cardiac output and an increased susceptibility to cardiac arrhythmias. It is postulated that electro-mechanical dyssynchrony may contribute to the susceptibility of the heart to cardiac arrhythmias. In this study, a novel system was developed to study these effects by altering the electro-mechanical activation sequence in cultured neonatal rat cardiomyocyte monolayers by dyssynchronously stimulating the monolayers with applied electrical fields and pulsatile mechanical strain. Specifically, optical mapping was utilized to compare action potential duration and quantify arrhythmia susceptibility of cardiomyocytes subjected to pulsatile mechanical strain, electrical stimulation, and dyssynchronous electrical and mechanical stimulation. This system provides a method to evaluate changes in cardiomyocyte conduction properties due to altered electro-mechanical coupling and the subsequent impact on arrhythmogenesis.
Assessment of Mechanical Cues to Enhance the Clinical Translation of Extracellular Vesicles
(2022) Kronstadt, Stephanie Marie; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Mesenchymal stem cells (MSCs) are a common source for cell-based therapies due to their innate regenerative properties. However, these cells often die shortly after injection and, if they do survive, run the risk of forming tumors. Cell-secreted nanoparticles known as extracellular vesicles (EVs) have been identified as having therapeutic effects similar to those of their parental cells without the safety risks. Specifically, MSC EVs have emerged as a promising therapeutic modality in a multitude of applications, including autoimmune and cardiovascular diseases, cancer, and wound healing. Despite this promise, low levels of naturally occurring EV cargo may necessitate repeated doses to achieve clinical benefit, countering the advantages of EVs over MSCs. The current techniques to combat low EV potency (e.g., loading external molecules or using chemicals) are not agreeable to large-scale manufacturing techniques and would substantially increase the regulatory burden associated with EV translation. Fortunately, mechanical cues within the microenvironment have potential to overcome these translational barriers as they can alter EV therapeutic effects but are also cost-effective and can be precisely manipulated in a reproducible manner. The goal of this project is to understand how these cues impact MSC EV secretion and physiological effects. We showed that flow-derived shear stress applied to MSCs seeded within a 3D-printed scaffold (i.e., the bioreactor) can significantly upregulate EV production (EVs/cell) while maintaining the in vitro pro-angiogenic effects of MSC EVs. Interestingly, we demonstrated that MSC EVs generated using the bioreactor system significantly improved wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Furthermore, for the first time, we showed that mechanical confinement of MSCs within micropillars could augment MSC EV production and bioactivity. Lastly, we demonstrated that soft substrates composed of various polydimethylsiloxane (PDMS) formulations could increase MSC EV production and activity as well. Through the work performed here, we have laid the groundwork to elucidate the relationship between cell mechanobiology and EV activity that will ultimately enable an adaptable and scalable EV therapeutic platform.
Auditory Streaming: Behavior, Physiology, and Modeling
(2011) Ma, Ling; Shamma, Shihab A; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Auditory streaming is a fundamental aspect of auditory perception. It refers to the ability to parse mixed acoustic events into meaningful streams where each stream is assumed to originate from a separate source. Despite wide interest and increasing scientific investigations over the last decade, the neural mechanisms underlying streaming still remain largely unknown. A simple example of this mystery concerns the streaming of simple tone sequences, and the general assumption that separation along the tonotopic axis is sufficient for stream segregation. However, this dissertation research casts doubt on the validity of this assumption. First, behavioral measures of auditory streaming in ferrets prove that they can be used as an animal model to study auditory streaming. Second, responses from neurons in the primary auditory cortex (A1) of ferrets show that spectral components that are well-separated in frequency produce comparably segregated responses along the tonotopic axis, no matter whether presented synchronously or consecutively, despite the substantial differences in their streaming percepts when measured psychoacoustically in humans. These results argue against the notion that tonotopic separation per se is a sufficient neural correlate of stream segregation. Thirdly, comparing responses during behavior to those during the passive condition, the temporal correlations of spiking activity between neurons belonging to the same stream display an increased correlation, while responses among neurons belonging to different streams become less correlated. Rapid task-related plasticity of neural receptive fields shows a pattern that is consistent with the changes in correlation. Taken together these results indicate that temporal coherence is a plausible neural correlate of auditory streaming. Finally, inspired by the above biological findings, we propose a computational model of auditory scene analysis, which uses temporal coherence as the primary criterion for predicting stream formation. The promising results of this dissertation research significantly advance our understanding of auditory streaming and perception.
Automated quantification and classification of human kidney microstructures obtained by optical coherence tomography
(2009) Li, Qian; Chen, Yu; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Optical coherence tomography (OCT) is a rapidly emerging imaging modality that can non-invasively provide cross-sectional, high-resolution images of tissue morphology such as kidney in situ and in real-time. Because the viability of a donor kidney is closely correlated with its tubular morphology, and a large amount of image datasets are expected when using OCT to scan the entire kidney, it is necessary to develop automated image analysis methods to quantify the spatially-resolved morphometric parameters such as tubular diameter, and to classify various microstructures. In this study, we imaged the human kidney in vitro, quantified the diameters of hollow structures such as blood vessels and uriniferous tubules, and classified those structures automatically. The quantification accuracy was validated. This work can enable studies to determine the clinical utility of OCT for kidney imaging, as well as studies to evaluate kidney morphology as a biomarker for assessing kidney's viability prior to transplantation.
Bio-templated Substrates for Biosensor Applications
(2013) Fu, Angela Li-Hui; Kofinas, Peter; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Nanopatterning of materials is of particular interest for applications in biosensors, microfluidics, and drug delivery devices. In biosensor applications there is a need for rapid, low cost, and durable system for detection. This dissertation aims to investigate methods to pattern nanostructured surfaces using virus particles as templates. The virus species used in these experiments is a cysteine modified tobacco mosaic virus. The first project utilized the lamellar microphase separation of a block copolymer to pattern the virus particles. Although microphase separation of the poly(styrene-b-2-vinylpyridine) (PS-P2VP) into lamellae was confirmed, specificity of the viruses to the gold doped block of the polymer could not be achieved. Single virus particles lay across multiple lamellae and aggregated in side-to-side and head-to-tail arrangements. The second project studied the effect of a surfactant on virus assembly onto a gold chip. The experiments included placing a gold chip in virus solutions with varying triton concentrations (0-0.15%), then plating the virus particles with a metal. Results showed that as the triton concentration in the virus solution increases, the virus density on the surface decreases. The gold coated virus particles were applied to Surface Enhanced Raman Spectroscopy (SERS) detection in the final project. SERS is of interest for biosensor applications due to its rapid detection, low cost, portability, and label-free characteristics. In recent years, it has shown signal enhancement using gold, silver, and copper nanoparticles in solutions and on roughened surfaces. The gold plated virus surfaces were tested as SERS substrates using R6G dye as the analyte. An enhancement factor (EF) of 10^4 was seen in these samples versus the non-SERS substrate. This corresponded to the sample with 0.05% triton in the virus solution which showed the most intersection points between the virus particles and the most uniform coverage of the viruses on the surface. This value is lower than that of previous studies; however, future work may be performed to optimize conditions to achieve the highest signal possible.
Biodegradable Prussian blue nanoparticles for photothermal immunotherapy of advanced cancers
(2015) Cano-Mejia, Juliana; Fernandes, Rohan; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Multifunctional nanoparticles represent a class of materials with diverse therapy and imaging properties that can be exploited for the treatment of cancers that have significantly progressed or advanced, which are associated with a poor patient prognosis. Here, we describe the use of biodegradable Prussian blue nanoparticles (PBNPs) in combination with anti-CTLA-4 checkpoint blockade immunotherapy for the treatment of advanced cancers. Our nanoparticle synthesis scheme yields PBNPs that possess pH-dependent intratumoral stability and photothermal therapy (PTT) properties, and degrade under mildly alkaline conditions mimicking the blood and lymph. Studies using PBNPs for PTT in a mouse model of neuroblastoma, a hard-to-treat cancer, demonstrate that PTT causes rapid reduction of tumor burden and growth rates, but results in incomplete responses to therapy and tumor relapse. Studies to elucidate the underlying immunological responses demonstrate that PTT causes increased tumor infiltration of lymphocytes and T cells and a systemic activation of T cells against re-exposed tumor cells in a subset of treated mice. PBNP-based PTT in combination with anti-CTLA-4 immunotherapy results in complete tumor regression and long-term survival in 55.5% of neuroblastoma tumor-bearing mice compared to only 12.5% survival in mice treated with anti-CTLA-4 alone and 0% survival both in mice treated with PTT alone, or remaining untreated. Further, all of the combination therapy-treated mice exhibit protection against tumor rechallenge indicating the development of antitumor immunity as a consequence of therapy. Our studies indicate the immense potential of our combination photothermal immunotherapy in improving the prognosis and outlook for patients with advanced cancers.
Bioelectronic Sensor for Cellular Assays Using Polyelectrolyte Multilayer-Modified Electrodes
(2008-04-25) Mijares, Geraldine; DeVoe, Donald L; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Cell-based impedance biosensors provide non-invasive, quantitative, and instantaneous detection of cellular responses to applied stimuli. Extracellular matrix proteins, which degrade over time, are commonly used as cell adhesion promoters on planar electrodes, but decrease the lifetime of biosensors. In this work, the feasibility of using non-biological polyelectrolyte multilayers (PEMs) to facilitate cell attachment on titanium-tungsten alloy/gold electrodes for cell assays is investigated. The PEMs-modified electrode system is modeled as an equivalent electrical circuit and the addition of cells to the system is defined by their electrical properties. Electrode performance is characterized by cyclic voltammetry and impedance spectroscopy. The electrodes are found to have the ability to specifically probe non-faradaic processes and show a 15% increase in impedance due to cell proliferation. This thesis work demonstrates the use of PEMs-modified electrodes for the continuous monitoring of cell proliferation and for the future application of probing cell confluency in microfluidic cytotoxicity assays.
Bioengineered conduits for directing digitized molecular-based information
(2015) Terrell, Jessica Lynn; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Molecular recognition is a prevalent quality in natural biological environments: molecules- small as well as macro- enable dynamic response by instilling functionality and communicating information about the system. The accession and interpretation of this rich molecular information leads to context about the system. Moreover, molecular complexity, both in terms of chemical structure and diversity, permits information to be represented with high capacity. Thus, an opportunity exists to assign molecules as chemical portrayals of natural, non-natural, and even non-biological data. Further, their associated upstream, downstream, and regulatory pathways could be commandeered for the purpose of data processing and transmission. This thesis emphasizes molecules that serve as units of information, the processing of which elucidates context. The project first strategizes a biocompatible assembly process that integrates biological componentry in an organized configuration for molecular transfer (e.g. from a cell to a receptor). Next, we have explored the use of DNA for its potential to store data in richer, digital forms. Binary data is embedded within a gene for storage inside a cell carrier and is selectively conveyed. Successively, a catalytic relay is developed to transduce similar data from sequence-based DNA storage to a delineated chemical cue that programs cellular phenotype. Finally, these cell populations are used as mobile information processing units that independently seek and collectively categorize the information, which is fed back as fluorescently ‘binned’ output. Every development demonstrates a transduction process of molecular data that involves input acquisition, refinement, and output interpretation. Overall, by equipping biomimetic networks with molecular-driven performance, their interactions serve as conduits of information flow.
Biological Nanofactories: Altering Cellular Response via Localized Synthesis and Delivery
(2008-11-19) Fernandes, Rohan; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Conventional research in targeted delivery of molecules-of-interest involves either packaging of the molecules-of-interest within a delivery mechanism or pre-synthesis of an inactive prodrug that is converted to the molecule-of-interest in the vicinity of the targeted area. Biological nanofactories provide an alternative approach to targeted delivery by locally synthesizing and delivering the molecules-of-interest at surface of the targeted cells. The machinery for synthesis and delivery is derived from the targeted cells themselves. Biological nanofactories are nano-dimensioned and are comprised of multiple functional modules. At the most basic level, a biological nanofactory consists of a cell targeting module and a synthesis module. When deployed, a biological nanofactory binds to the targeted cell surface and locally synthesizes and delivers molecules-of-interest thus altering the response of the targeted cells. In this dissertation, biological nanofactories for the localized synthesis and delivery of the 'universal' quorum sensing signaling molecule autoinducer-2 are demonstrated. Quorum sensing is process by which bacterial co-ordinate their activities at a population level through the production, release, sensing and uptake of signaling autoinducers and plays a role in diverse bacterial phenomena such as bacterial pathogenicity, biofilm formation and bioluminescence. Two types of biological nanofactories; magnetic nanofactories and antibody nanofactories are presented in this dissertation as demonstrations of the biological nanofactory approach to targeted delivery. Magnetic nanofactories consist of the AI-2 biosynthesis enzymes attached to functionalized chitosan-mag nanoparticles. Assembly of these nanofactories involves synthesis of the chitosan-mag nanoparticles and subsequent assembly of the AI-2 pathway enzymes onto the particles. Antibody nanofactories consist of the AI-2 biosynthesis enzymes self assembled onto the targeting antibody. Assembly of these nanofactories involves creation of a fusion protein that attaches to the targeting antibody. When added to cultures of quorum sensing bacteria, the nanofactories bind to the surface of the targeted cells via the targeting module and locally synthesize and deliver AI-2 there via the synthesis module. The cells sense and uptake the AI-2 and alter their natural response. Prospects of using biological nanofactories to alter the native response of targeted cells to a 'desired' state, especially with respect to down-regulating undesirable co-ordinated bacterial response, are envisioned.
BIOMATERIALS REPROGRAM ANTIGEN PRESENTING CELLS TO PROMOTE ANTIGEN-SPECIFIC TOLERANCE IN AUTOIMMUNITY
(2023) Eppler, Haleigh B; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The immune system is tightly regulated to balance the killing of disease-causing organisms while protecting host tissue from accidental damage. When this balance is disrupted, immune dysfunctions such as autoimmune diseases occur. Autoimmune diseases like type 1 diabetes and multiple sclerosis (MS) develop when self-tissue is mistakenly attacked and damaged by immune cells. For example, during MS, the immune system mistakenly attacks the myelin sheath that insulates neurons, causing loss of motor function and burdening patients and caregivers. Recent advances in immunotherapies offer exciting new treatments; however, even monoclonal antibody therapies cannot differentiate between healthy and disease-causing cells. Biomaterials provide powerful capabilities to help address these shortcomings. In particular, control over the concentration, duration, location, and combination of signals that are received by immune cells could be transformative in developing more selective immunotherapies that are safe and promote antigen-specific tolerance during autoimmune disease. This dissertation uses two biomaterial approaches to deliver regulatory cargo to antigen presenting cells (APCs). An important APC function is to detect disease-causing organisms by sensing pathogen associated molecular patterns (PAMP) through motif-specific receptors. CpG rich motifs are PAMPs that activate toll-like receptor 9 (TLR9) on DCs and B cells. TLR9 signaling activates B cells and DCs. In MS, TLR9 signaling is aberrantly elevated on certain DCs contributing to systemic inflammation. In MS, B cells signaling through the TLR9 pathwway induced the expression of more inflammatory cytokines as compared to B cells from healthy controls. Controlling this overactive TLR signaling restrains inflammation and is a possible tolerogenic therapeutic approach in MS. The first part of this dissertation uses biomaterials-based polyelectrolyte multilayers (PEMs) to deliver tunable amounts of GpG – an oligonucleotide that inhibits TLR9 signaling – to dendritic cells (DCs). These studies demonstrate that PEMs inhibit DC activation and reduce pathway-specific inflammatory signaling. Furthermore, this work demonstrates that these changes to DCs promote tolerance in downstream T cell development as shown by increasing regulatory T cells. These studies demonstrate this biomaterial delivery system selectively inhibits TLR signaling and DC activation. These changes to DCs promote myelin-specific T cells to adopt a regulatory phenotype, demonstrating a potential approach to developing tolerance inducing antigen-specific immunotherapies for MS. The second part of this dissertation uses a degradable polymer microparticle (MP) system to control the local microenvironment of lymph nodes (LNs). LNs are key sites in the development of immune responses. LNs are composed of different microdomains that coordinate immune cell interactions such as germinal centers (GCs), where B cells develop. These MPs are loaded with myelin self-antigen (MOG35-55) and an mTOR inhibitor, rapamycin (rapa). The MPs are designed to be too large to passively diffuse from the LNs; instead, they slowly degrade releasing encapsulated immune cues to immune cells within the lymph node (LN). Our previous work demonstrates this treatment approach induces antigen-specific tolerance in a preclinical model of MS, but the role of APCs – including DCs and B cells - has not been elucidated. This dissertation reveals that MP treatment alters key LN structural components responsible for interactions between cells in GCs. In addition, MPs alter interactions between B cells/DCs and T cells, as measured by presentation of encapsulated antigen and inhibition of T cell costimulatory molecules by encapsulated rapa. These changes inhibit myelin-specific T cell proliferation and promote regulatory T cells. Finally, B cells from MOG/rapa and MOG MP treated lymph nodes transfer myelin-specific efficacy to mice induced with EAE. These findings illustrate how LN and cellular processes can be regulated by MPs to promote myelin-specific tolerance informing the development of myelin-specific immunotherapies for MS. Together, this body of work provides insight into how biomaterials can be designed to exploit native LN and immune cell functions in the design of next-generation approaches to safely induce myelin-specific tolerance during MS or other autoimmune diseases.
BIOMIMETIC NANOSTRUCTURES FOR THERANOSTIC APPLICATIONS
(2015) Kuo, Yuan-Chia; D'Souza, Warren D; Raghavan, Srinivasa R; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Theranostic nanostructures are those that have both therapeutic as well as diagnostic function, e.g., due to having a combination of drugs as well as imaging agents in them. Such structures, especially those that can selectively home in on cancer tumors, have received considerable attention recently. Although many different structures have been synthesized, their complexity, high cost, and poor biocompatibility have limited their clinical application. In this study, we focus on creating new classes of theranostic nanostructures using simple routes (via self-assembly) and utilizing inexpensive and biocompatible materials. In our first study, we describe a class of liposomal probes that can allow certain tumors to be imaged by magnetic resonance imaging (MRI). Tumors, such as those of head and neck cancer, are known to over-express the epidermal growth factor receptor (EGFR). Our liposomal probes bear anti-EGFR antibodies as well as chelated gadolinium (Gd), a positive (image-brightening) contrast agent for MRI. To synthesize these probes, we use a strategy that is carefully designed to be simple and scalable: it employs two steps that each involve self-assembly. The resulting probes bind in vitro to EGFR-overexpressing tumor cells compared to controls. Moreover, cancer cells with bound probes can be tracked by MRI. In the future, these probes could find clinical use for tracking the efficacy of cancer treatment in real-time. Next, we report a class of microscale (3 to 5 µm) containers derived from erythrocytes (red blood cells). Micro-erythrosomes (MERs) are produced by emptying the inner contents of these cells (specifically hemoglobin) and resuspending the empty structures in buffer. We have developed procedures to functionalize the surfaces of the MERs with targeting moieties (such as anti-EGFR antibodies) and also to load solutes (such as fluorescent dyes and MRI contrast agents) into the cores of the MERs. Thus, we show that MERs are a versatile class of microparticles for biomedical applications. In our final study, we show that the MERs from the previous study can be sonicated to yield nanoscale structures, termed nano-erythrosomes (NERs), with average sizes around 120 nm. NERs are membrane-covered nanoscale containers, much like liposomes. They show excellent colloidal stability in both buffer as well as in serum at room temperature, and they are able to withstand freeze-thaw cycling. Moreover, NER membranes can be decorated with fluorescent markers and antibodies, solutes can be encapsulated in the cores of the NERs, and NERs can be targeted towards mammalian cells. Thus, NERs are a promising and versatile class of nanostructures for use in nanomedicine.
Biophysical Aspects of Leukocyte Transmigration through the Vascular Endothelium
(2011) Stroka, Kimberly Murley; Aranda-Espinoza, Helim; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Leukocyte transmigration through the vascular endothelium is a key step in the immune response, and also in progression of the cardiovascular disease atherosclerosis. Much work has previously focused on the biological aspects of leukocyte transmigration, such as cytokine exposure, junctional protein organization in the endothelium, and signaling pathways. However, in recent years, many studies have identified links between the mechanical properties of the cellular microenvironment and cell behavior. This is relevant to the cardiovascular system in two ways: (1) it is likely that the mechanical properties of vasculature depend on both vessel size (large vessels versus microvasculature) and tissue type (soft brain versus stiffer muscle or tumor), and (2) both large vessels and microvasculature stiffen in atherosclerosis. For the first time, this dissertation provides a quantitative evaluation of the biophysical effects of vasculature stiffening on endothelial cell (EC) biomechanical properties, as well as leukocyte migration and transmigration. A novel in vitro model of the vascular endothelium was created. This model mimics physiological conditions more closely than previous models, by taking into account the flexibility of the subendothelial matrix; previous models have mostly utilized glass or plastic substrates that are much stiffer than physiological. EC monolayers were formed on extracellular matrix (ECM) protein-coated hydrogels and activated with tumor necrosis factor-α or oxidized low density lipoprotein to induce an inflammatory response. We determined that three important components of the in vitro model (cell-cell adhesion, cytokine exposure, and subendothelial matrix stiffness) have significant effects on EC biomechanical properties. Next, we showed that neutrophils are mechanosensitive, as their migration is biphasic with substrate stiffness and depends on an interplay between substrate stiffness and ECM protein amount; these results suggest that any biomechanical changes which occur in vasculature may also affect the immune response. Finally, we discovered that neutrophil transmigration increases with subendothelial matrix stiffness, and we demonstrated that this effect is due to substrate stiffness-dependent EC contractile forces. These results indicate, for the first time, that the biophysical states of the endothelium and subendothelial matrix, which likely vary depending on size, location, and health of vasculature, are important regulators of the immune response.
A biophysical evaluation of cell-substrate interactions during spreading, migration and neuron differentiation
(2010) Norman, Leann Lynn; Aranda-Espinoza, Helim; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The development of engineered scaffolds has become a popular current avenue to treat numerous traumas and disease. In order to optimize the efficiency of these treatments, it is necessary to have a more thorough understanding of how cells interact with their substrate and how these interactions directly affect cellular behavior. Cell spreading is a critical component of numerous biological phenomena, including embryonic development, cancer metastasis, immune response, and wound healing. Along with spreading, cell adhesion and migration are all strongly dependent on the interactions between the cell and its substrate. Cell-substrate interactions can affect critical cellular mechanisms including internal cellular signaling, protein synthesis, differentiation, and replication and also influence the magnitude of adherence and motility. In an effort to better understand cell-substrate interactions we characterize the initial stages of cell spreading and blebbing using cell-substrate specific microscopy techniques, and identify the effects of cytoskeletal disruption and membrane modification on surface interactions and spreading. We identify that blebs appear after a sharp change in cellular tension, such as following rapid cell-substrate detachment with trypsin. An increased lag phase of spreading appears with increased blebbing; however, blebbing can be tuned by supplying the cell with more time to perform plasma membrane recycling. We developed software algorithms to detect individual bleb dynamics from TIRF and IRM images, and characterize three types of bleb-adhesion behaviors. Overall, we show that blebs initially create the first adhesion sites for the cell during spreading; however, their continuous protrusion and retraction events contribute to the slow spreading period prior to fast growth. In addition, we identify the elastic modulus of the rat cortex and characterize a polyacrylamide gel system that evaluates the effects of substrate stiffness on cortical outgrowth. Remarkably, we illustrate that cortical neuron differentiation and outgrowth are insensitive to substrate stiffness, and observe only morphological differences between laminin versus PDL-coated substrates. Together, this research identifies cell-specific behaviors critical to spreading and migration. The thorough evaluations of spreading and migration behavior presented here contribute to the understanding of critical cellular phenomena and suggest potential therapeutic targets for treatment of cardiovascular disease and neurological disorders.
Blood Coagulation Inducing Synthetic Polymer Hydrogel
(2010) Casey, Brendan John; Kofinas, Peter; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Uncontrolled hemorrhaging, or blood loss, accounts for upwards of 3 million deaths each year and is the leading cause of preventable deaths after hospital admission around the world. Biological-based hemostatics are quite effective at controlling blood loss, but prohibitively expensive for people in developing countries where over 90 % of these deaths are occurring. Synthetic-based hemostatics are less expensive, yet not nearly as effective as their biological counterparts. A better understanding of how synthetic materials interact with and affect the body's natural clotting response is vital to the development of future hemostatic material technology which will help millions around the world. Initial in vitro experimentation focused on investigating the key chemical and structural material properties which affect Factor VII (FVII) activation in citrated human plasma. Enzyme-linked assays were utilized to confirm the ability of specifically formulated charged hydrogels to induce FVII activation and provided insight into the critical material parameters involved in this activation. Dynamic mechanical analysis was used to establish a correlation between polymeric microstructure and FVII activation. Experiments utilizing coagulation factor depleted and inhibited plasmas indicated that FVII, FX, FII, and FI are all vital to the process outlining the general mechanism of fibrin formation from the onset of FVII activation. The ability of the polymer to induce fibrin formation in "artificial plasma" explicitly lacking calcium, TF, and platelets suggested that a specifically designed material surface has the capability to substitute for these vital cofactors. Clinical diagnostic experimentation using sheep blood indicated that hydrogels containing higher amounts of electrostatic positive charge and lower cross-link density were able to induce faster, more robust clot formation in the presence of a coagulation cascade activator. Subsequent in vivo animal experimentation clearly demonstrated the ability of such hydrogels to aggregate platelets and erythrocytes promoting the formation of an effective hemostatic seal at the wound site. Moreover, in vivo testing confirmed the viability of such a charged polymer hydrogel to effectively control blood loss in a clinically relevant model.

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